DEV Community: EDITGENE

Prime Editing--Precise gene editing

EDITGENE — Sun, 04 Jan 2026 08:50:14 +0000

Gene editing is a precise modification of the genome, which is an important means of studying gene function. At present, the commonly used gene editing method is the CRISPR-Cas9 system. After a decade of development, the CRISPR-Cas9 system has made breakthroughs in gRNA, PAM, single base editing, and multi bases editing. However, it is still difficult to achieve arbitrary modification of bases.
In 2019, Professor David Liu's team from Harvard University developed a method for precise gene editing based on CRISPR - Prime editing. In theory, Prime editing can achieve the editing effects of all editing methods today.

Prime editing is a technology optimized on the basis of traditional CRISPR/Cas9. Prime editing consists of two parts: one is nCas9 and reverse transcriptase fusion protein, and the other is pegRNA (prime editing gRNA). Unlike general gRNAs, pegRNA not only targets to specific DNA region that needs to be edited, but also carrying a "reverse transcription template".

Cas9-reverse transcriptase fusion protein will precisely cleave the target DNA single strand under the guidance of pegRNA, and then synthesize DNA containing the corrected bases according to the "reverse transcription template". The DNA repair mechanism within cells will integrate this newly synthesized sequence into the genome.

Prime Editing principle

Prime Editing is capable of performing highly specified deletion, insertion, and base exchange. The biggest difference from other CRISPR-based technologies is that it does not generate DNA double strand breaks (DBS) and does not require donor DNA, making it very easy to achieve precise editing.

Advantages of Prime Editing
(1) High efficiency: More efficient than HDR, editing efficiency reach to 30-40%.
(2) Various types of editing: point mutation, insertion, and precise knockout.
(3) Low off-target effect.

With over a decade of experience in gene editing, EDITGENE has developed the Bingo™ platform based on cutting-edge Prime Editing (PE) technology This platform integrates highly efficient sgRNA design with a rigorous monoclonal screening system, enabling the customized generation of various types of precise point mutation cell lines for our customers.

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Decoding DNA: From the Double Helix to the Central Dogma

EDITGENE — Fri, 26 Dec 2025 08:51:28 +0000

After nearly a century of contemplation and verification by scientists, it was confirmed: DNA is the hereditary material.

However, new questions emerged: Why DNA? How can such a microscopic molecule carry genetic information so vast and precise?

DNA is composed of only four simple deoxyribonucleotide molecules, yet it must describe the entire spectrum of an organism's traits. It is akin to asking a pen that can only write the letters "A, B, C, and D" to compose the epic masterpiece War and Peace. The mystery, far from being solved, only grew more profound.

01.The Emergence of the Double Helix: Structure Reveals the Secrets of Information

The history of science is never short of turning points.

In 1953, a magical configuration leaped out from the blurry shadows of X-rays: the DNA double helix structure. James Watson and Francis Crick drew inspiration from the X-ray diffraction patterns of DNA crystals captured by crystallographers Maurice Wilkins and Rosalind Franklin, successfully constructing the mysterious yet elegant double helix model.

Two strands intertwined, with "letters" pairing in the simplest possible way: A with T, and C with G. Like buttons. Like a key finding its lock. Like a pair of dance partners who never miss a step. It is precisely these base-pairing rules that allow DNA to structurally carry and replicate information.

While observing the model, Watson proposed a bold hypothesis: DNA replicates in a semiconservative manner. The two original strands unwind, with each being passed to one of the two offspring, subsequently generating two new strands identical to the parent. But how could this be proven? And why must DNA adopt this "half-old, half-new" mode of replication?

Figure 1. The semiconservative DNA replication model

02.Truth Under Density Gradient: Semiconservative Replication

In 1958, Matthew Meselson and Franklin Stahl provided the answer. They designed and executed what would later be hailed as one of "the most beautiful experiments in biology."

Building upon the ingenious design of the Hershey-Chase experiment, they utilized the weight differences between isotopes to culture bacteria. After growing several generations in a medium containing the N-15 isotope, they transferred the bacteria to a medium containing N-14. From that point on, any DNA replication could only utilize the N-14 isotope.

By comparing the density of the DNA molecules across generations, Meselson and Stahl reached the only logical conclusion: In every cycle of division, the DNA in the daughter bacteria consists of a double helix formed by one parental strand and one newly synthesized strand. Semiconservative replication thus received irrefutable experimental evidence.

Figure 2. The Meselson-Stahl experiment

At this point, the clues of a century were finally connected: From Mendel’s peas to Griffith’s mice; From bacteriophage infections to the mysterious "X" captured by X-rays; These fragmented clues, like a vast net, were drawn tight through successive experiments, finally capturing the "figure" hidden in the depths of life: DNA.

03.The Next Challenge: From Base Sequences to Biological Traits

However, a new question followed: "How does the sequence of bases in DNA determine traits?" Once genetic information is written into DNA, how is it "read" by the cell? The answer lay in another class of molecules that had once been overlooked by early geneticists: Proteins.

Compared to DNA, which is composed of only four simple nucleotides, proteins are far more complex. Proteins possess unpredictable three-dimensional structures; they can fold, bend, and catalyze chemical reactions. Our understanding of proteins predates our understanding of DNA by many years..

While Watson and Crick were building the DNA model, their colleagues Max Perutz and John Kendrew were attempting to analyze the 3D structure of protein molecules. Success for these two scientists came a bit later; in 1959, they successfully resolved the 3D structure of hemoglobin, revealing for the first time the high degree of protein complexity..

This discovery was timely. Only after DNA was confirmed as the carrier of genetic information did it make sense to ask: How does the relatively simple DNA guide the cell in synthesizing such a diverse and functional array of proteins?

04.Deciphering the Code

Surprisingly, the first logical step toward solving this puzzle was taken on scratch paper rather than in a petri dish.

Physicist George Gamow, a key figure in the Big Bang theory, speculated that the four bases must be organized in a specific pattern to correspond to the 20 amino acids in proteins.

If two bases corresponded to one amino acid (4²), the combinations were insufficient.

If four bases corresponded to one amino acid (4⁴), it seemed redundant.

Only three bases (4³=64) provided just enough coding space.

In 1961, Marshall Nirenberg and Johann Matthaei cracked the first codon: UUU → Phenylalanine. However, strictly speaking, their experiment only proved that DNA sequences corresponded to amino acid sequences; it did not prove the exact number of bases per amino acid.

Har Gobind Khorana provided the solution. Using more complex long-chain nucleic acid sequences, he proved that only a triplet sequence could correspond to a single amino acid. Over the next five years, scientists from various institutions worked together to decode all combinations: 64 codons corresponding to 20 amino acids—an unbelievably elegant design.

Figure 3. The central dogma

Now, returning to that yellow pea, we can finally trace the entire process in reverse: The pea contains a protein that determines the formation of its surface pigment. The sequence of amino acids for this protein is written in the pea’s DNA, with three bases corresponding to each amino acid.

DNA does not directly participate in protein synthesis. In the cell, genetic information must first be "transcribed" into an intermediate molecule—RNA. RNA then "translates" the base sequence into an amino acid sequence to synthesize a protein.

DNA stores information, RNA translates it, and proteins execute it..

This is the Central Dogma. The Central Dogma is like the main melody of life’s operation; every cell sings according to this rhythm. By understanding this pathway, humanity can finally explain the origin of hereditary traits, identify the roots of genetic diseases, and begin to envision precise, limited interventions within this flow of information.

Life may be vast and complex, but its underlying logic has finally been understood, written, and gradually mastered by humankind. And the exploration of genes continues.

At EDITGENE, our custom gene knockout cell line services utilize an advanced CRISPR/Cas9 system to support research teams in bridging basic research and clinical applications. Feel free to reach out anytime to design a gene editing plan tailored to your research needs.

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At Just One Year Old, He Joins Nature’s 10: The Story of a CRISPR Breakthrough

EDITGENE — Mon, 22 Dec 2025 09:55:00 +0000

KJ Muldoon

The Youngest Entry in the History of "Nature’s 10"

In the 2025 edition of Nature’s 10—the annual list of ten people who helped shape science—an unprecedented name appeared: a one-year-old infant.

He was not chosen for conducting research, but for being the subject of a medical milestone that redefined the boundaries of gene editing and therapy. His name is KJ Muldoon.

Figure 1. KJ Muldoon (Source: Nature)

Reports indicate that KJ Muldoon is the first patient in the world to successfully receive a personalized in vivo CRISPR gene-editing therapy. This breakthrough is considered a pivotal step in moving gene editing from the laboratory into real-world clinical applications.

Previously, there have been pioneering cases in personalized gene therapy. The most well-known was Mila, whose treatment (Milasen) was custom-designed for a specific mutation in her genome. While it did not fully cure her, it successfully slowed the progression of her disease. This attempt laid the foundation for personalized medicine and is widely regarded as a milestone for N-of-1 (single-patient) gene therapy.

Following Mila, other patients have achieved significant results through gene editing, but most of those cases involved ex vivo editing (where cells are edited outside the body and then re-infused).

KJ’s treatment, however, represents a radical leap: it is the world’s first in vivo personalized CRISPR therapy. Scientists encapsulated the gene-editing tools within lipid nanoparticles (LNPs) and injected them directly into KJ’s bloodstream. These particles specifically targeted liver cells to perform DNA repair. Theoretically, this means KJ may only require a limited number of treatments to achieve a permanent cure.

01.A Fatal Challenge: A Countdown from Birth
Just 48 hours after birth, KJ began exhibiting symptoms of lethargy and respiratory distress.

Blood tests revealed a staggering blood ammonia concentration of 1,703 μmol/L—more than 50 times the normal range. Such levels are typically seen only in severe urea cycle disorders or acute liver failure.

In healthy individuals, blood ammonia levels range from 9-33 μmol/L; as ammonia accumulates, it causes neurological damage, including lethargy and seizures. Further elevation leads to coma, cerebral edema, and death.

Figure 2. Schematic of the urea cycle

The urea cycle is a critical metabolic pathway in the liver. Its primary function is to convert toxic ammonia (NH₃), a byproduct of protein breakdown, into non-toxic urea, which is then excreted via the kidneys. This process safely removes excess nitrogen and maintains nitrogen balance.

Pediatricians at the Children’s Hospital of Philadelphia (CHOP) immediately initiated Continuous Renal Replacement Therapy (CRRT) and ordered genetic testing.

The results showed two nonsense mutations in KJ’s CPS1 gene: c.1003C→T and c.2140G→T, inherited from each parent. KJ was formally diagnosed with Carbamoyl Phosphate Synthetase 1 (CPS1) deficiency. This is an extremely rare genetic disorder with an estimated incidence of less than one in a million. Although individual rare diseases are scarce, they collectively affect millions worldwide, often with high mortality rates and little hope for survival.

Conventional treatments, such as low-protein diets and liver transplantation, were not viable options for KJ at the time, as his infant body could not withstand the high risks of a transplant surgery.

02.Initiating Treatment: Applying Personalized CRISPR Therapy
Faced with KJ’s deteriorating condition, pediatricians and scientists collaborated to attempt a tailor-made solution using cutting-edge CRISPR technology.

The research team utilized Base Editing, a derivative of CRISPR genome editing. This technology allows for the precise correction of a mutation by swapping a single DNA base pair. By avoiding the double-strand breaks used in traditional CRISPR, it minimizes the risk of indel errors and reduces off-target effects.

Figure 3. Base Editing

The team targeted the Q335X mutation (c.1003C→T) inherited from KJ’s father. Using an Adenine Base Editor (ABE), they targeted the A-T base pair at the mutation site and converted it into a normal G-C base pair. This restored the correct amino acid sequence and, consequently, the function of the CPS1 gene.

To ensure the editing tools reached the liver accurately, researchers used lipid nanoparticles (LNPs) as delivery vehicles. The CRISPR editors were encapsulated within these LNPs and administered to KJ via intravenous injection.

This highly customized therapy, named "k-abe," became the world’s first personalized CRISPR gene-editing therapy successfully applied to an infant.

03.Significant Outcomes: KJ’s Road to Recovery
• At ~1 month old: A patient-specific cell line was established to study KJ’s unique genetic sequence.

• At ~6 months old: As his condition worsened, KJ was officially placed on the liver transplant waiting list.

Simultaneously:

• Researchers worked around the clock to manufacture the gene-editing components in just six months—a process that normally takes 18 months.

• The team conducted comprehensive testing, including toxicology studies in non-human primates and mice.

• The team submitted a “Compassionate Use” application to the FDA; it was approved within a week, allowing the treatment to proceed.

On February 25, 2025—209 days after birth—KJ received the world’s first personalized in vivo CRISPR treatment. The LNPs entered his circulation, were taken up by hepatocytes, and successfully repaired the pathogenic mutation in the CPS1 gene.

Since then, KJ’s blood ammonia levels have not seen a significant spike. He has been able to gradually increase his protein intake, and his vital signs remain stable. While it is too early to declare a "total cure," the marked improvement in the early stages of treatment has brought new hope for his future.

As of April 2025, KJ has received three doses of the therapy with no serious side effects. Shortly after treatment, he began tolerating increased protein intake and required fewer nitrogen scavengers.

In May 2025, a report from the National Institutes of Health (NIH) referred to KJ as the "first infant to successfully receive personalized gene therapy." His treatment was meticulously designed to target non-germline cells, ensuring the genetic changes would not be passed to future generations.

Figure 4. NIH related report

04.The Future of Personalized Gene Therapy
KJ’s success has sparked widespread interest in the medical community regarding personalized gene therapy.

His story is more than a personal victory over a life-threatening illness; it represents a massive leap in the application of genome engineering. It showcases the vast potential of CRISPR in personalized medicine, especially for addressing various rare genetic diseases.

Through personalized therapy, CRISPR has evolved from a laboratory concept into a viable clinical solution. The U.S. FDA recently announced an accelerated approval pathway for personalized treatments for rare genetic diseases, opening new doors for clinical applications.

Challenges remain, including long-term safety, the stability of therapeutic effects, and the high cost of customized treatments. Is KJ’s "N=1" miracle a prologue to the widespread adoption of gene therapy, or a unique case difficult to replicate?

The answer will be written by time and continued scientific endeavor.

05.References:
1.Ledford, H. (2025). World’s first personalized CRISPR therapy given to baby with genetic disease. Nature, News Feature.

2.Ledford, H. (2025). The baby whose life was saved by the first personalized CRISPR therapy. Nature, News Feature.

3.National Center for Advancing Translational Sciences (NCATS). (2025). Infant with rare, incurable disease is first to successfully receive personalized gene therapy treatment. National Institutes of Health (NIH).

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Peas, Bacteria, and DNA: A Century-Long Pursuit of Genetic Truth

EDITGENE — Wed, 17 Dec 2025 07:38:38 +0000

It all began with a single pea plant.

Humanity’s earliest experiments in heredity happened unconsciously, in sheep pens and wheat fields—transplanted grains that happened to thrive, short-legged sheep that conveniently stayed inside fences.

People selected from whatever traits emerged in each generation, but success came only through long waits and countless failures, arriving more by accident than by understanding.

When they tried to combine a “fiery-tempered meat hog” with a “small, gentle piglet” in hopes of creating the perfect animal, what they got instead were pigs that were both scrawny and irritable—one disappointing outcome after another.

People could see the results, but the pattern behind them remained invisible. They had no idea why good traits could vanish without warning, or why undesirable ones kept returning as if mocking their efforts.

01.The Science That Began in a Monastery Garden

Around this time, a lean, quiet priest—Gregor Mendel—planted a collection of pea plants in the backyard of St. Thomas Abbey, tucked away on the outskirts of the Austro-Hungarian Empire.

He wanted to push humanity’s long, confusing tradition of trial-and-error breeding away from “divine mystery” and toward something that could finally be explained by science.

Mendel set aside every established theory and returned to the facts. He gathered data—mountains of it—and tried to make sense of the patterns hidden within.

He chose traits that were measurable, visible, and cleanly distinct: tall vs. short, yellow vs. green, purple vs. white.

From their repeated patterns he forged a new idea—dominant and recessive traits.

Over eight years, and through more than eight thousand cross-bred pea seeds, Mendel discovered something remarkable:

green peas reappeared in the second generation; the same 3:1 ratio of traits surfaced again and again, as if obeying an unseen rule.

These patterns revealed a radical truth: heredity wasn’t a matter of fluids blending and fading.

It was built from “particles”—independent units that could separate and recombine in every new generation.

Figure 1. Results of the second pea cross

The ways in which genetic information can combine are virtually endless, yet the units themselves are remarkably resilient and stable. They can lie dormant for generations, only to reappear when the conditions are right.

In a sense, Mendel provided a solid, material foundation for Darwin’s theory, while Darwin gave Mendel’s discoveries a grand stage on which to demonstrate their significance.

02.What Exactly Are These “Particles” of Inheritance?

At first, Mendel simply called them “heritable factors.”

It wasn’t until the 20th century, when scientists dusted off Mendel’s old manuscripts, that his theory of particulate inheritance was rediscovered—and these heritable factors were given a new name: genes.

If Mendel’s peas in the monastery garden allowed humanity to glimpse the rules of inheritance for the first time, the generations of scientists that followed were determined to grasp the very essence of the gene.

They approached life from a chemical perspective. If genes were indeed particles, then surely they could be isolated and extracted, like any other substance.

They asked themselves: “If we could extract a particular substance from a yellow pea and introduce it into a green one to make it yellow, would this substance be the ‘yellow gene’?”

And so began a true chase for the gene itself—a quest that would reshape our understanding of life at the molecular level.

03.A Dead Mouse Reveals a Crucial Clue

Fred Griffith, while studying Streptococcus pneumoniae, stumbled upon a surprising clue.

He injected mice with a mixture of heat-killed “smooth” bacteria and live “rough” bacteria—a procedure that should have been the safest, most mundane experiment imaginable.

Yet the mouse died.

Even more puzzling, live smooth bacteria were found inside the dead mouse.

One mouse, one clue.

Griffith didn’t fully understand what had happened, but the result suggested something astonishing:

“Some form of ‘information’ had passed from the dead bacteria to the living ones, transforming them.”

Figure 2. Griffith’s pneumococcal transformation experiment

Oswald Theodore Avery at the Rockefeller Institute picked up the trail where Griffith left off.

He boiled smooth pneumococcal bacteria and systematically stripped away their components—lipids, proteins, polysaccharides—one layer at a time.

In the end, only a clear, fibrous substance remained.

This fibrous molecule had the chemical composition of DNA.

And crucially, the transformation effect disappeared only when enzymes that specifically degraded DNA were added.

Avery concluded that DNA was the substance carrying hereditary information.

From our vantage point today, we can easily cheer for Avery’s breakthrough.

He provided the sharpest explanation yet for the particulate inheritance Mendel had proposed nearly a century earlier.

But in the 1940s, the scientific community wasn’t ready to accept it.

“Perhaps,” they said,“you simply didn’t remove the proteins thoroughly enough.”

04.Viruses Deliver the Final Answer

After Thomas Hunt Morgan demonstrated through fruit fly experiments that genes reside on chromosomes, DNA once again surfaced in connection with heredity.

Coincidence—again?

American scientists Alfred Hershey and Martha Chase provided a decisive answer, using viruses as their tool.

They designed an elegant experiment: label DNA and proteins with different radioactive isotopes, let viruses infect bacteria, and then track which substance appears in the next generation of viruses.

The result was unmistakable:

it was the DNA that entered the offspring, not the protein.

At last, the true identity of the gene emerged.

Figure 3. The Hershey-Chase Experiment

05.The End of a Century-Long Pursuit, and the Start of Something New

From Mendel’s tiny particles of heredity, to Griffith’s perplexing results, to the confirmations by Avery, Hershey, and Chase, humanity spent nearly a century chasing down the truth:

“It is a strand of DNA.”

“It passes from one organism to another, determining traits.”

“It is the carrier of life’s blueprint.”

Yet this realization was not an ending.

On the contrary—it placed us at the true threshold of modern biology.

From this point on, the story of the gene finally began.

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How to utilize CRISPR library to speed up your research?

EDITGENE — Tue, 16 Dec 2025 05:57:32 +0000

With the rapid development of CRISPR/Cas9 gene editing technology, CRISPR library screening has played an important role in drug screening, viral infection, and tumor functional gene screening experiments. Compared with cDNA libraries and RNAi libraries, CRISPR library screening has the advantages of versatility, low noise, high knockout efficiency, and low off-target rate, making it a popular method for candidate gene screening.

Q: What is CRISPR library screening?
A: CRISPR library screening is a method for accurate screening for functional genes.

Q: What’s the difference between two plasmid library and one plasmid library？
One plasmid system: It includes promoter, gRNA, Cas9, and selection marker. gRNA and Cas9 can be simultaneously transferred into cells. However, due to the large size of the one plasmid system, its infection efficiency is low. Two plasmid system: only carries gRNA and the promoter; Cas9 stable cell line generation is needed. The two plasmid system has higher efficiency and is more convenient to modify the plasmid, but requires two transductions. In this article, we will introduce the procedure and tips of two-plasmid system CRISPR library screening. Let’s get started now!

01.Procedure

1.Generation of Cas9 stable cell line
1) Cas9 lentivirus packaging;
2) Lentivirus transduction and Cas9 expressing stable cell clones isolation and screening;
3) Cas9 activity test.

2. sgRNA library construction (pre-made or customized)
Design 3-6 sgRNAs for each gene, then each sgRNA clones to a plasmid backbone, and then amplify the plasmid library and NGS sequencing.

3. sgRNA library lentivirus packaging
1) Mix sgRNA library and helper plasmids and transfect 293T cells in T225 culture flask;
2) After 48 hours, collect culture media and store in -80℃.

4. Lentivirus transduction (take A549 as an example)
1) Test for A549 kill curve, and determine the optimal concentration of puromycin for screening;
2) Transduce 2×10^7 A549-Cas9 cells with 0.3 MOI (i.e. 6×10^6 cells are transduced), each sgRNA in this library integrates to >1000 cells.

5. Cell phenotype screening (take A549 as an example)
Divide the whole genome knockout cell pool into two parts.
1) One is the experimental group to exert screening pressure, such as virus infection, drug treatment, etc;
2) The other one is the control group. Screening cells based on phenotypes such as drug resistance, proliferation ability, and survival ability.
Note: There are positive screening and negative screening according to different screening purposes. Generally 3 control groups and 3 experimental groups are needed to lower the statistic error.

6.NGS sequencing and data analysis
Perform the following steps on the experimental group and the control group:
1) Genome extraction; 2) PCR amplification of sgRNAs;
3) NGS sequencing;
4) Bioinformatics analysis (MaGCKFlute).

7. Gene function study
Validate gene function from two aspects: the necessity and sufficiency.
1) Knockout each candidate gene;
2) Overexpression or rescue experiment.

02.Tips

The keys to successful CRISPR library screening:
1) Selecting a high cutting efficiency Cas9 stable cells is the key to obtain a KO cell library with high coverage rate.
2) Screening range: for high-throughout screening, the size of a library is one of the important aspects. Thus, try to choose small libraries and prioritize pre-made libraries.
3) During lentivirus transduction, ensure one lentivirus particle enter one cell.

EDITGENE provides one-stop CRISPR library screening service. We provide over 200 types of Cas9 stable cell lines, pre-made human/mouse libraries, customized library service. Let us do the job, and provide you the trustworthy data!

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How Did We Edit Genes Before CRISPR?

EDITGENE — Tue, 09 Dec 2025 06:51:10 +0000

Since the discovery of the DNA double-helix structure in the 1950s, scientists have gradually recognized that alterations in gene sequences can influence cellular functions, paving the way for the exploration of gene-editing technologies. Broadly speaking, any intentional modification to an endogenous gene sequence—such as knockout, insertion, replacement, or disruption of regulatory regions—can be considered gene editing.

With advances in sequencing technologies and molecular genetics, it became increasingly clear that many important biological phenotypes and even diseases arise from subtle changes in genetic sequences. Single-nucleotide substitutions, or point mutations, are among the most common forms of genetic variation, involving the alteration of a single nucleotide or nucleotide pair within DNA. These mutations may occur spontaneously or be induced by environmental factors, and can influence protein function or gene expression, resulting in synonymous, missense, or nonsense mutations.

Among the tens of thousands of identified disease-associated variants, single-nucleotide substitutions account for the largest proportion. This has driven the development of more precise gene-editing tools that allow researchers to model, correct, or investigate these mutations with greater accuracy-advancing basic research, disease-mechanism studies, and drug-target validation.

Figure 1. Overview of common gene-editing approaches

Early gene-editing technologies evolved from relying on natural cellular mechanisms to employing engineered nucleases. In this section, we highlight three pivotal approaches: gene targeting, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). These methods laid the groundwork for modern gene editing and significantly advanced research in genetics, developmental biology, and biomedical science.

01.Gene Targeting: The First Milestone in Mammalian Gene Editing

Gene targeting is an early gene-engineering strategy that enables precise modification of genomic DNA. By designing a DNA fragment homologous to the target gene—often carrying specific edits or selectable markers—researchers can leverage the cell’s own homologous recombination machinery to insert the exogenous DNA at the intended locus.

▌ Technical Principle
The core mechanism behind gene targeting is homologous recombination—a natural DNA repair process in which cells use a matching DNA template to repair double-strand breaks. Researchers design a targeting vector containing two “homology arms” identical to the sequences flanking the desired genomic locus, with the intended insertion or replacement—such as selectable markers or reporter genes—placed in between.

The gene-targeting workflow generally involves two major steps:

Gene editing in mouse embryonic stem (ES) cells
A targeting vector is constructed and introduced into ES cells using electroporation. Through homologous recombination, precise modifications can be introduced into the ES-cell genome.
Generation of gene-modified mice
ES cells carrying the desired mutation are selected and microinjected into mouse blastocysts. After chimeric mice are born, germline transmission is confirmed through breeding, completing the gene-targeting process.

Gene targeting opened the era of mammalian gene editing and laid the technological and conceptual foundation for many gene-editing tools that followed. Several fundamental laboratory techniques developed alongside gene targeting—such as stem-cell isolation, culture, and characterization, electroporation-based transfection, and microinjection—remain widely used in research today.

02.Zinc Finger Nuclease Technology (ZFN):The First Generation of Engineered Gene Editing

As the first generation of engineered gene-editing technology, zinc finger nucleases (ZFNs) were discovered and developed in the late 1990s by Aaron Klug’s laboratory.

This technology fuses the DNA-binding domains of zinc finger proteins with the cleavage domain of the FokI endonuclease, creating an artificial chimeric nuclease.

ZFNs induce site-specific double-strand breaks in cellular DNA, significantly enhancing the efficiency of homologous recombination-mediated repair. This effectively overcomes the low efficiency and limited applicability of traditional gene-targeting approaches.

▌ Technical Principle
The core mechanism of ZFNs is “recognition and cleavage”—specific DNA sequence recognition followed by targeted double-strand break (DSB) formation.

Figure 2. Schematic representation of Zinc Finger structure

DNA Recognition Domain: Zinc Finger Arrays
Zinc finger proteins are common transcription factor domains stabilized by a zinc ion coordinated with multiple amino acids, such as cysteine and histidine, forming “finger-like” structures. Each “finger” can specifically recognize a triplet of nucleotides. By linking 3-6 zinc fingers in tandem, a zinc finger array capable of recognizing a 9-18 base pair sequence is created, enabling precise targeting of a specific genomic locus.
DNA Cleavage Domain: FokI Endonuclease
FokI is a restriction endonuclease derived from Flavobacterium okeanokoites. In ZFNs, only its cleavage domain is retained, without any inherent DNA sequence recognition capability.
Dimerization and DNA Cleavage
ZFNs must be designed and applied as pairs, each targeting sequences upstream and downstream of the intended DNA site. When both ZFN molecules bind to their respective target sites, the two FokI cleavage domains are brought into proximity, dimerize, and become activated. They cleave the DNA in the intervening region (typically 5-7 base pairs apart), generating a double-strand break.

The cell then repairs the break via non-homologous end joining (NHEJ) or homology-directed repair (HDR), inducing site-specific recombination that can result in gene knockout, knock-in, or correction.

▌ Clinical Breakthroughs
ZFN technology has also made significant strides in clinical applications. In November 2017, the first human clinical trial assessing the safety and tolerability of in vivo genome editing using ZFNs commenced, targeting patients with mucopolysaccharidosis I (MPS I), MPS II, and hemophilia B. The study demonstrated successful genome editing in vivo and, in some patients, transiently increased enzyme activity to approximately half of normal levels. Despite its limitations, ZFN paved the way for the development of subsequent gene-editing technologies.

03.Transcription Activator-Like Effector Nucleases (TALEN):The Second-Generation Modular Gene-Editing Tool

Following ZFNs, TALENs (Transcription Activator-Like Effector Nucleases) represent the second generation of engineered gene-editing technologies. Their core principle involves fusing TALE proteins—derived from the plant pathogen Xanthomonas, capable of recognizing specific DNA sequences—with the FokI nuclease responsible for DNA cleavage. This fusion enables precise double-strand breaks at targeted genomic loci, subsequently inducing gene knockout, knock-in, or repair. Compared to ZFNs, TALENs offer greater flexibility in sequence design and improved targeting specificity, making them easier to use in practice.

Figure 3. Schematic representation of TALEN structure

▌ Technical Principle
The core of TALENs lies in the TALE protein, which consists of three main components: a nuclear localization signal, a transcriptional activation domain, and a DNA-binding domain. The DNA-binding domain is composed of multiple tandem repeat units, each recognizing a single nucleotide. The amino acids at positions 12 and 13 of each repeat—known as the repeat-variable di-residue (RVD)—determine the nucleotide recognition specificity.

In TALENs, the TALE DNA-binding domain is fused to the FokI nuclease cleavage domain, creating an artificial nuclease. Only when a pair of TALENs binds to adjacent target sequences does the FokI domain dimerize, become activated, and cleave the DNA to generate a double-strand break (DSB). The cell then repairs the break through non-homologous end joining (NHEJ) or homology-directed repair (HDR), resulting in gene knockout or precise knock-in.

04.Comparison of Advantages and Disadvantages

TALEN technology, with its modular protein design, achieved a revolutionary decoupling of DNA recognition and cleavage functions, marking a major leap in gene-editing specificity, flexibility, and safety. It effectively overcame many limitations of ZFN technology by employing programmable DNA-binding domains, greatly expanding the targeting range while significantly reducing off-target effects and cytotoxicity, thereby enabling nearly any genomic sequence to be edited.

The successful application of TALENs not only demonstrated the broad feasibility of efficient and precise gene editing but also provided critical design principles and practical experience that informed the development and optimization of subsequent, more streamlined, and powerful tools such as CRISPR.

EDITGENE focuses on CRISPR technology, offering a comprehensive range of high-quality gene-editing services and in vitro diagnostic products. These include CRISPR library screening, cellular gene editing and CRISPR detection, among others. We are dedicated to providing the most efficient technical support for scientific research related to CRISPR, gene function studies, in vitro diagnostics, and therapeutic applications.

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Gene Editing: From Field-Grown Wheat to Precision Genomes

EDITGENE — Tue, 09 Dec 2025 06:22:22 +0000

If time could speak, it would probably find humans quite amusing, for we often get things right first and only later figure out why.

Thousands of years ago, humans had no concept of genes. There was no DNA, no mutations, no Cas9. Yet somehow, everyone was engaged in the same endeavor—unconsciously harnessing natural variation, selecting traits from the chaos of nature.

Take, for example, that wheat that would not shatter its ears. In the wild, it was destined to die out, a casualty of nature’s ruthless selection.

But to humans, this “non-shattering” trait was a boon, making harvest far easier. They transplanted it around their villages, nurtured it carefully, and awaited the first mature seeds.
Bit by bit, this mutation—highly disadvantageous to the wheat itself, yet profoundly beneficial to humans—was preserved.

This was perhaps the earliest form of “artificial selection,” and with it, agriculture truly began to take root.

Figure 1. Wild-type teosinte (left) and today’s widely cultivated maize crop (right)

The two hardly look like they belong to the same species. During the domestication of maize, the change in ear size was even more remarkable.

At that time, people could not explain such phenomena, and so the gods naturally became the default answer.

But in more rational Greece, thinkers like Democritus and Hippocrates began to seek scientific explanations for why traits could be inherited. They proposed the concept of pangenesis, an attempt to give this “gift of the gods” a rational basis—the theory of blending particles.

This idea persisted into modern times, providing a conceptual foundation for Darwin, who formulated his theory of evolution. Darwin imagined that traits mixed and transmitted through mating much like pigments blending together.

Yet doubts quickly arose: if inheritance truly worked like ink dropped into milk, then even the most pronounced variations would be rapidly diluted and vanish. In that case, the cumulative changes that evolution relies upon could never occur.

In an era utterly ignorant of genes, conflicting voices clashed relentlessly, striving to unravel the true nature of inheritance.

Figure 2. The contradiction between the theory of pangenesis (left) and the theory of natural selection (right)

According to the theory of pangenesis, tiny variations in the parents’ gemmules would be “diluted” during reproduction and thus disappear. This stands in conflict with the theory of natural selection, which holds that even small hereditary variations can be passed on— forming the material basis of natural selection.

Time seemed to sit back and watch all of this unfold, as if observing a millennium-long debate play out on a distant stage.

Inheritance operated in darkness, and humanity made its choices in that same darkness.

It was not until the rise of modern genetics that a light was finally switched on, revealing the true logic behind that ancient “non-shattering wheat.”

In reality, humans had been making use of genetic variation for thousands of years — long before we understood what we were actually doing.

For a very long time, we were doing the right things without knowing why.

Today, when we talk about CRISPR, people call it “ the scalpel of God. ”

Yet this history quietly reminds us that gene editing did not descend out of nowhere; it is the natural extension of centuries of observation, trial, and accumulated experience.

We did not suddenly acquire this “scalpel.”
We merely came to understand how it works — moving from intuitive selection to precise editing.

At EDITGENE, our custom gene knockout cell line services utilize an advanced CRISPR/Cas9 system to support research teams in bridging basic research and clinical applications. Feel free to reach out anytime to design a gene editing plan tailored to your research needs.

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From CRISPR Screening to FCD Mechanisms: Do Cilia Also Influence Nerves?

EDITGENE — Tue, 02 Dec 2025 08:11:42 +0000

Cilia protrude from the surface of most cells in the body and play a crucial role in cellular signaling. Research has shown that defects in ciliary assembly can lead to "ciliopathies" in humans, characterized by multisystem developmental disorders. Additionally, cilia are typically resorbed prior to cell growth and differentiation to maintain normal cellular function. However, our understanding of the regulatory mechanisms underlying the critical process of regulated ciliary disassembly remains limited.

On October 29, 2025, David K. Breslow's team at Yale University published a research paper titled "A CRISPR activation screen reveals a cilia disassembly pathway mutated in focal cortical dysplasia" in Science Advances. This study employed a genome-wide CRISPR library screening strategy, utilizing the CRISPRa system to construct an sgRNA library targeting 22,774 genes, to systematically identify regulators that promote ciliary disassembly.

Original link: https://www.science.org/doi/10.1126/sciadv.aeb7238

Spotlight

Innovative Screening Strategy: CRISPRa Technology Uncovers Negative Regulators of Cilia
The research team established a mouse NIH-3T3 ciliary cell line expressing dCas9-VP64 and conducted a genome-wide CRISPRa screen using a Blasticidin selection reporter system dependent on the Hedgehog (Hh) signaling pathway. By overexpressing 22,774 mouse genes, key ciliary disassembly regulators such as F2R and SARM1 were identified.
Mechanistic Breakthrough: Discovery of a SARM1-Mediated Calcium-Dependent Ciliary Disassembly Pathway
The study revealed a SARM1-mediated calcium-dependent ciliary disassembly pathway: thrombin activates the receptor F2R, leading to the production of the second messenger cADPR by SARM1, which activates RyR to induce calcium release from the endoplasmic reticulum. The localized increase in calcium subsequently triggers the RhoA/ROCK pathway, driving ciliary disassembly.
Disease Relevance: Gene Mutations in the Ciliary Disassembly Pathway Linked to Various FCD Subtypes
The study found that mutations in genes such as SARM1, RYR2/3, and RHOA in patients with Focal Cortical Dysplasia (FCD) lead to excessive ciliary disassembly and impaired cortical neuron development. Although the pathogenic genes differ across FCD subtypes, they all rely on the SARM1 pathway to mediate ciliary disassembly, highlighting its role as a common key mechanism in FCD pathogenesis.

01.Identification and Validation of F2R and SARM1 in Inducing Ciliary Disassembly

Through a genome-wide CRISPRa screen (covering 22,774 genes), the research team identified F2R and SARM1 as key regulators of ciliary disassembly. Functional validation demonstrated that overexpression of either gene significantly reduced cilia, while their specific inhibitors (vorapaxar and DSRM-3716) completely rescued ciliary loss.

Subsequently, the team conducted doxycycline (Dox)-induced F2R overexpression experiments, confirming that F2R can trigger disassembly of pre-existing cilia.

Figure 1. Overexpression of F2R or SARM1 Induces Ciliary Disassembly

02.Elucidation of Ciliary Disassembly Mechanisms and Subcellular Localization

The study further delineated the mechanism and subcellular localization of F2R–SARM1-mediated ciliary disassembly.

Experiments revealed that SARM1 acts downstream of F2R activation. Moreover, using inhibitors such as dantrolene (an RyR inhibitor) and the calcium chelator BAPTA-AM, it was found that cADPR produced by SARM1 activates RyR channels, leading to calcium release. This subsequently triggers ciliary disassembly via RhoA-targeted activation of ROCK kinase.

Figure 2. SARM1-Triggered Signaling Pathway Mediates Ciliary Disassembly

03.Validation of Endogenous Regulation

During serum- or lysophosphatidic acid (LPA)-induced ciliary disassembly, inhibition of SARM1, RyR, or ROCK significantly preserved ciliary stability. This confirms that the F2R–SARM1–RyR–RhoA/ROCK pathway is an endogenous key pathway mediating serum mitogen-induced ciliary disassembly.

Figure 3. Endogenous Regulation of Ciliary Dynamics by the SARM1-Driven Pathway

04.Therapeutic Potential of Targeting the Pathway in FCD

The study revealed that FCD patients carry mutations in genes such as SARM1, RYR2/3, and RHOA, which overlap with this ciliary disassembly pathway. These mutations were found to cause abnormal development of cortical neurons.

Subsequently, the research team constructed disease models including SARM1-G528S, RhoA-P75S, and mTORC1-activating mutants to simulate the pathogenic mechanisms of different FCD subtypes. The results demonstrated that inhibiting the SARM1/RyR/RhoA pathway exhibited therapeutic efficacy against both Type I and Type II FCD.

Figure 4. Components of the Ciliary Disassembly Pathway Are Mutated in FCD

05.Summary

This study, through CRISPRa screening, identified a complete ciliary disassembly signaling pathway: F2R → SARM1 → cADPR → RyR → calcium ions → RhoA. More importantly, it establishes the first link between abnormalities in the ciliary disassembly pathway and the pathogenesis of Focal Cortical Dysplasia (FCD), highlighting the therapeutic potential of targeting this pathway in FCD.

Future research should aim to further elucidate the connection mechanism between F2R/LPAR1 and SARM1, delineate the molecular details of how RhoA/ROCK drives ciliary disassembly, and validate the prevalence and pathological contribution of ciliary defects in FCD patient tissues.

EDITGENE offers comprehensive end-to-end solutions for CRISPR library screening, from custom sgRNA library design and stable Cas9 cell line development to lentiviral library packaging, library cell pool establishment, functional screening experiments, and NGS data analysis. We provide a wide selection of popular pre-designed libraries and most extensive collection of ready-to-ship library plasmids and library viruses —all available within one week. Place your order and start screening immediately!

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Knocking Out the HO-1 Gene Makes Tumor Cells More

EDITGENE — Fri, 28 Nov 2025 07:30:24 +0000

Photodynamic therapy (PDT) induces immunogenic cell death (ICD) mediated by reactive oxygen species (ROS), leading to the release of endogenous tumor-associated antigens, neoantigens, and damage-associated molecular patterns. As a non-invasive in situ therapy, PDT has been shown to trigger the generation of an autologous vaccine. However, ROS not only reduces ICD yield but may also contribute to tumor recurrence and distant metastasis as potential seed cells. Therefore, eliminating tumor resistance to ROS and achieving heritable ROS sensitivity is crucial.

Recently, a collaborative study by Sichuan University and the National University of Singapore published in Nature Biomedical Engineering titled "A HO-1 gene knockout using a NanoCRISPR scaffold suppresses metastasis in mouse models" reported that their designed Nano CRISPR scaffold (a nanocarrier-based CRISPR gene editing system) achieved a gene editing efficiency of 20.15%, without compromising the gene editing capabilities or cytotoxic functions of key immune cells. By knocking out the HO-1 gene , the Nano CRISPR system transformed the heterogeneous phenotype of tumor cells into a ROS-sensitive state, thereby enhancing the efficacy of ICD and improving the outcomes of in situ vaccination.

Original link: https://doi.org/10.1038/s41551-025-01518-1

Spotlight

ROS Susceptibility
The Nano CRISPR system achieves permanent knockout of HO-1, effectively enhancing tumor susceptibility to ROS without adversely affecting key immune cells.
Superior Therapeutic Efficacy
Under near-infrared (NIR) irradiation, the in situ autologous nano-vaccine (AVAX) treatment group demonstrated a tumor growth inhibition rate of 93%, significantly outperforming the AVAX-Ce6+L group (83%).
Potent Immunological Properties
When combined with laser irradiation and anti-PD-L1 antibody therapy, the Nano CRISPR platform induces robust antitumor immunity and long-term immunological memory in melanoma models.

01.Design and Validation of AVAX
Photodynamic therapy (PDT) induces immunogenic cell death (ICD) mediated by reactive oxygen species (ROS), leading to the release of endogenous tumor-associated antigens, neoantigens, and damage-associated molecular patterns. As a non-invasive in situ therapy, PDT has been shown to trigger the generation of an autologous vaccine. However, ROS not only reduces ICD yield but may also contribute to tumor recurrence and distant metastasis as potential seed cells. Therefore, eliminating tumor resistance to ROS and achieving heritable ROS sensitivity is crucial.

The designed AVAX completely releases CpG and Ce6 in the presence of hyaluronidase and in weakly acidic environments, demonstrating its ability to elicit a hierarchical response to both the intra- and extracellular tumor microenvironment.

Figure 1. Design and Characterization of the AVAX Nanovaccine

02.AVAX Overcomes ROS Tolerance
AVAX enables efficient delivery of the CRISPR-Cas9 system into tumor cells and achieves targeted knockout of HO-1, a key gene identified for ROS tolerance.

In B16F10 melanoma and LL/2 lung cancer models, a single intravenous injection combined with NIR irradiation resulted in a 20.15% HO-1 gene editing efficiency in tumor cells. This enhanced PDT efficacy, induced an autologous vaccine effect, and activated long-lasting antitumor immune responses. This strategy permanently alters tumor cell sensitivity to ROS through genetic modification.

Figure 2. AVAX Overcomes Tumor Resistance to Reactive Oxygen Species

03.Immune Activation and Biodistribution of AVAX In Vitro
NIR-irradiated AVAX enhances ICD in tumor cells, subsequently activating bone marrow-derived dendritic cells (BMDCs) and potentially promoting antigen presentation.

Moreover, AVAX was predominantly taken up by tumor cells in vivo, facilitating HO-1 knockout, while showing minimal impact on BMDCs, T cells, and macrophages, with no significant toxicity observed.

Figure 3. ICD Activation by AVAX and Its Preferential Uptake in Tumors

04.Antitumor Efficacy of AVAX In Vivo
Under identical NIR irradiation conditions, the AVAX treatment group achieved a 93% tumor growth inhibition rate, significantly outperforming the AVAX-Ce6+L group (83%) and other treatment groups that showed no notable tumor suppression.

Additionally, ROS accumulation in the AVAX group was markedly higher than in other comparative groups. These results collectively demonstrate the excellent antitumor efficacy of AVAX in vivo.

Figure 4. Enhanced Tumor Suppression and ROS Accumulation Mediated by AVAX In Vivo

05.Prevention of Tumor Recurrence
In primary tumor resection experiments, the combination of AVAX with L&αPD-L1 significantly reduced tumor recurrence rates, with 5 out of 6 experimental mice maintaining a tumor-free survival period exceeding 75 days. In rechallenge experiments, this group exhibited the slowest tumor growth, confirming that the combined therapy effectively suppresses recurrence and induces durable immune memory.

Figure 5. AVAX Combined with L&αPD-L1 Significantly Suppresses Tumor Recurrence and Induces Immune Memory

Summary
This study successfully reversed the genetic tolerance of tumor cells to ROS through a core–shell structured Nano CRISPR system, achieving "genetic sensitization" of tumors. The approach inhibited tumor growth by up to 93% in vivo and significantly suppressed tumor recurrence when combined with L&αPD-L1 therapy.

This strategy opens new avenues for personalized tumor immunotherapy. Future efforts may focus on optimizing delivery systems and safety profiles, integrating gene editing with immune activation mechanisms to develop more efficient and precise therapeutic solutions for tumors.

EDITGENE specializes in providing customized knockout cell line as its core service. Leveraging an advanced CRISPR/Cas9 system, we consistently equip research teams with key tools to bridge the gap between basic research and clinical applications. Contact us today to customize your gene-editing solutions and accelerate your project!

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sgRNA Design and Screening

EDITGENE — Thu, 27 Nov 2025 10:43:44 +0000

To achieve efficient gene point mutation and knock-in, it is essential to design sgRNAs with strong cleavage activity and high HDR efficiency.

The design of sgRNAs requires a comprehensive evaluation of multiple factors — including the location of candidate editing sites, GC content, and potential off-target risks — to select one or more sgRNAs with high cleavage efficiency and specificity for experimental validation.

The following principles can be used as guidelines for sgRNA design :

01.General Design Principles for Conventional sgRNAs
✅ GC content should preferably not be lower than 40%
✅ Avoid four consecutive T bases within the target sequence
✅ Perform specificity analysis to reduce nonspecific off-target mutations

02.Minimize Distance Between Cleavage Site and Editing Site
To ensure high homologous recombination efficiency, the insertion or point mutation site should be located as close as possible to the sgRNA cleavage site. Existing studies have shown a clear monotonic negative correlation between editing efficiency and distance. In general, the ideal distance is within 10 bp. Beyond this range, recombination efficiency drops significantly.

Figure 1. Editing efficiency exhibits a monotonically negative correlation with distance

03.Design Multiple sgRNAs for Parallel Screening
High theoretical scores do not always guarantee optimal performance in actual cellular experiments.

We recommend initially screening 2–4 candidate sgRNAs in parallel to identify the most efficient one for subsequet experiments.

04.Off-Target Risk Assessment
sgRNA guides the Cas9 enzyme to the target genomic site via base complementarity. However, sequence homology elsewhere in the genome can cause Cas9 to cleave at non-target sites, resulting in off-target effects and undesired mutations.

To minimize off-target risks, the following strategies can be applied:

① Optimize sgRNA Design:
This is the most fundamental step. As the cornerstone of specificity, sgRNAs should be designed using professional software to ensure high genome-wide specific, optimal GC content and avoiding consecutive repetitive sequences.

② Use High-Fidelity Cas9 Variants
Several high-fidelity versions of Cas9 (e.g., SpCas9-HF1, eSpCas9) have been developed. These variants require more precise sgRNA-DNA pairing, significantly reducing mismatch tolerance and effectively mitigating off-target effects.

③ Improve Delivery Methods and Control Expression Duration
Transient delivery methods, such as direct introduction of ribonucleoprotein complexes (RNPs), rather than using plasmids for sustained expression, can shorten the exposure time of Cas9/sgRNA in cells and lower the chance of unintended cleavage.

④ Apply Comprehensive Off-Target Detection Techniques
Technologies like GUIDE-seq and OGM provide genome-wide off-target assessment, offering a realistic profile of gene editing safety.

Leveraging our proprietary sgRNA design logic, along with advanced synthesis and purification technologies, EDITGENE offers professional sgRNA design and synthesis services to support enterprises and research institutions in efficiently advancing their gene editing projects.

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Rewrite DNA, Precisely — With CRISPR

EDITGENE — Thu, 20 Nov 2025 07:02:34 +0000

I.Mechanism of the CRISPR/Cas9 System
At the core of the CRISPR/Cas9 system lies its ability to use a sgRNA to specifically recognize a target DNA sequence and direct the Cas9 endonuclease to introduce a double-strand break (DSB) upstream of the PAM (Protospacer Adjacent Motif) sequence.
This site-specific DNA cleavage triggers the cell’s intrinsic DNA repair machinery, primarily through two pathways: non-homologous end joining (NHEJ) and homology-directed repair (HDR).

✅ NHEJ repair often results in small insertions or deletions (indels), leading to gene disruption—a mechanism widely used in gene knockout) applications.
✅ HDR repair, on the other hand, utilizes an exogenous DNA template to precisely repair the break, forming the foundation for point mutation and gene knock-in experiments.

Image sourced online. Copyright belongs to the original creator

II.Image sourced online. Copyright belongs to the original creator
Homology-directed repair (HDR) is a high-fidelity DNA double-strand break (DSB) repair mechanism naturally present in cells. It uses a homologous DNA sequence as a template for precise repair and serves as the core technology for gene knock-in and point mutation cell line construction. The HDR process involves the following steps:

1. DSB Formation and Recognition.
Guided by the sgRNA, CRISPR-Cas9 introduces a double-strand break in the target DNA. The MRN complex recognizes the broken ends and initiates repair.

Image sourced online. Copyright belongs to the original creator

2. End Resection
The BRCA1/MRN/CtIP complex resects the 5′ ends of the DNA, generating 3′ single-stranded overhangs that provide directionality for HDR.

3. Single-Strand DNA Binding and RAD51 Replacement
RPA initially binds to the single-stranded DNA to prevent degradation. Subsequently, RAD51 replaces RPA to form a nucleoprotein filament, preparing for homology search.

4. Strand Invasion and DNA Synthesis
The RAD51 filament searches for a homologous sequence, and the 3′ single strand invades the template DNA, serving as a blueprint for DNA synthesis.

5. Repair Completion
The newly synthesized DNA is resolved through either double-strand break repair (DSBR) or synthesis-dependent strand annealing (SDSA) pathways, ultimately restoring genomic integrity.

Image sourced online. Copyright belongs to the original creator

III.Why Ideal HDR Is Often Less Efficient in Practice
Although HDR enables precise DNA repair, its actual efficiency is frequently limited by multiple factors:

✅ Competition with NHEJ: HDR competes with the faster and more prevalent non-homologous end joining (NHEJ) pathway, which often repairs DSBs before HDR can occur.
✅ Low delivery efficiency of donor DNA: Donor templates, such as ssODNs or double-stranded DNA, often enter the nucleus inefficiently, reducing the chances of HDR.
✅ Cell cycle dependence: HDR is predominantly active during the S/G2 phases of the cell cycle, restricting its window of opportunity.
✅ Cell type variability: Different cell types display varying sensitivities to transfection and DNA repair, further limiting HDR efficiency.

As a result, HDR has become synonymous with “high precision but low efficiency” in genome editing experiments.

IV.Strategies to Improve HDR Efficiency
To overcome these limitations, researchers have developed several optimization approaches:

✅ Inhibiting NHEJ: Small molecules such as SCR7 can block DNA-PKcs or Lig4 activity to reduce NHEJ competition.
✅ Cell cycle modulation: Drugs or synchronization methods can increase the proportion of cells in the S/G2 phase, where HDR is most active.
✅ Optimizing donor template delivery: Systems like AAV or LNPs can enhance nuclear uptake of donor DNA.
✅ Cas9 engineering: Using nickase or high-fidelity Cas9 variants can reduce off-target effects and erroneous repair.

By applying these strategies, HDR efficiency can be significantly increased in certain cell lines, providing a more robust approach for generating precise point mutations and gene knock-in models.

V.Conclusion
The combination of CRISPR/Cas9 and HDR has laid the foundation for precise genome editing. Although efficiency improvements are still needed, its potential continues to unfold, gradually enabling truly customized genetic modifications.

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4T1 Luciferase Stable Cell Line: Advancing Breast Cancer Metastasis Research

EDITGENE — Tue, 18 Nov 2025 10:24:38 +0000

The 4T1 Luciferase (4T1-Luc) stable cell line is a mouse breast cancer cell line engineered to stably express the Firefly Luciferase reporter gene, derived from the highly metastatic parental 4T1 line. Through bioluminescence imaging, the 4T1-Luc cell line enables real-time, non-invasive monitoring of tumor growth and metastasis, making it an ideal model for studies on breast cancer metastasis, drug screening, and immunotherapy.
This instruction provides a detailed overview of how to use the 4T1-Luc cell line to establish breast cancer metastasis models and apply them in scientific research, covering experimental protocols and optimization strategies. By using our ready-to-use 4T1-Luc product, researchers can rapidly initiate experiments and significantly reduce the time required for model construction.

I.Advantages of the 4T1-Luc Cell Line in Breast Cancer Research
The 4T1-Luc cell line, with its high metastatic potential and stable bioluminescent signal, has become a key tool in breast cancer research and is suitable for the following applications:

● Metastasis studies: Models lung, liver, and bone metastasis of triple-negative breast cancer, allowing real-time tracking of metastatic progression.
● Drug screening: High-sensitivity bioluminescence enables high-throughput screening of chemotherapeutics, targeted therapies, or nanomedicines.
● Immunotherapy research: Used in combination with CAR-T cells or PD-1 inhibitors to evaluate changes in the tumor immune microenvironment.
● Gene function validation: Supports gene knockout or overexpression (e.g., CRISPR-mediated E-cadherin editing) to study EMT pathways.
● In vivo and ex vivo modeling: Compatible with orthotopic mouse models; non-invasive IVIS imaging reduces the number of animals required.

II. Product Recommendation
The 4T1-Luc cell line provided by EDITGENE exhibits >99% purity and high activity, with bulk orders eligible for discounts.

Luciferase Assay Results:

Advantages:
● Purity >99%, activity >1.2 × 10⁶ RLU/10⁵ cells (n=3, SD <5%).
● Stable for >20 passages; metastasis efficiency >80% (validated by IVIS).
● COA and SOP are provided; discounts available for bulk orders.

III.Construction of 4T1-Luc Cell Line
Pinciple of Construction
The 4T1-Luc cell line is engineered using genetic modification techniques, where the luciferase (luc) gene is inserted into a suitable vector and transfected into the parental 4T1 cells to achieve stable expression. Luciferase catalyzes the oxidation of its substrate, luciferin, generating bioluminescence with a peak wavelength of approximately 550-570 nm. The luminescence intensity correlates linearly with enzyme expression levels. Common configurations include single-luciferase systems or dual-luciferase systems, the latter incorporating Renilla luciferase as an internal control for experimental normalization.

Common Construction Methods
The most widely used approach is lentiviral transduction, as it allows efficient and stable integration. The following outlines a typical workflow using EDITGENE’s 4T1-Luc cell line as an example:

1. Construct the expression vector:
a. Clone the luciferase (luc) gene into a lentiviral vector (e.g., pLenti or pLVX).
b. The vector typically includes a promoter (such as CMV) to drive luc expression, a selectable marker (e.g., puromycin resistance) for screening, and a fluorescent tag (e.g., GFP) for visualization.
c. For dual-luciferase systems, Renilla luciferase can be included as an internal control, either co-expressed or placed on the same plasmid (e.g., pGL3 or pmirGLO).

2.Lentivirus packaging:
a. Co-transfect the recombinant vector with helper plasmids (packaging plasmid psPAX2 and envelope plasmid pMD2.G) into HEK293T cells to produce lentiviral particles.
b. Collect the viral supernatant, filter, and determine titer (typically 10⁸-10⁹ TU/mL)

3. Transduce parental cells:
a. Select parental 4T1 mouse breast cancer cells and culture until 70-80% confluence.
b. Add viral particles at a multiplicity of infection (MOI) of 10-50 and incubate for 48-72 hours.
c. Transduction efficiency can be evaluated by GFP fluorescence or preliminary luciferase activity assay.

4. Select stable clones:
a. 24-48 hours post-transduction, add antibiotic (e.g., 2-5 μg/mL puromycin) to select for positive cells.
b. Continue selection for 7-14 days until surviving cells form single clones.
c. EDITGENE employs 3D single-cell printing technology to isolate single clones.
（3D single-cell printing precisely separates individual cells, greatly improving the accuracy of monoclonal screening and cell survival. This non-contact method avoids mechanical damage and background contamination, maintaining cell integrity and biological activity. Compared with traditional limiting dilution, 3D single-cell printing reduces human error and ensures reliable screening outcomes.）

5. Validation and Applications
a. Validation Methods: Gene integration can be confirmed by qPCR, protein expression by Western blot, and luciferase activity by bioluminescence assay.
b. Validation Criteria: Luciferase activity > 10⁶ RLU/10⁵ cells, with stable expression maintained for over 20 passages.
c. Applications: Cells can be injected into mouse models, and tumor growth can be monitored using an IVIS imaging system after intraperitoneal administration of the luciferase substrate.ction.

IV. Application Examples: Advancing Research Projects
4.1 Breast Cancer Metastasis Mechanism
● Objective: Investigate the role of the Wnt signaling pathway in lung metastasis.
● Method: Knockout the β-catenin gene, inject 4T1-Luc cells, and monitor metastatic changes using IVIS imaging.
● Result: Quantification of bioluminescence signals to assess metastasis rates, supporting mechanistic studies.

4.2 Drug Screening
● Objective: Screen novel PI3K inhibitors for breast cancer suppression.
● Method: Perform in vitro high-throughput screening and measure luminescence signals.
● Result: Rapid generation of IC50 curves, providing data for publications.

4.3 Immunotherapy
● Objective: Evaluate the efficacy of PD-L1 inhibitors in combination with nanocarriers.
● Method: Administer combination treatment in mouse models and monitor tumor burden via IVIS imaging.
● Result: Dynamic data support translational research.

V. Summary
The 4T1-Luc stable cell line is a powerful tool for constructing breast cancer metastasis models, playing a critical role in real-time tumor progression monitoring, drug screening, and immunotherapy research. Its high-sensitivity bioluminescence provides reliable data, accelerating research progress. Building this cell line in the laboratory can be time-consuming, whereas EDITGENE’s 4T1-Luc product offers optimized expression (>90% positive rate) and high metastatic efficiency, with COA and SOP included. We invite you to order our 4T1-Luc cells to kick-start your breakthrough research.

VI.References
● Tiscornia, G., et al. (2006). Nature Protocols, 1(1), 241–245. DOI: 10.1038/nprot.2006.37.
● Pulaski, B. A., & Ostrand-Rosenberg, S. (2001). Mouse 4T1 breast tumor model. Current Protocols in Immunology. DOI: 10.1002/0471142735.im2002s39.
● Kim, J. B., et al. (2004). Non-invasive monitoring of tumor growth with bioluminescence imaging. Cancer Research. DOI: 10.1158/0008-5472.CAN-04-1234.

The Luciferase cell line provided by EDITGENE is stably expressing firefly luciferase. This cell line was tested for luciferase activity. Useful for in vitro and in vivo imaging. EDITGENE has a large inventory of Luciferase cells and can provide high-quality products in an efficiently and timely.Luc Overepression Cell Line Construction

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